Recent work from the principal investigator's laboratory and others has uncovered a super-family of ubiquitin E3 ligases containing a common dimeric core element formed by combinatorial interactions between the ROC/APC11 RING-H2 finger protein and cullin (CUL)/APC2 family proteins. Members among this E3 ligase class include SCF-ROC1, Anaphase-Promoting Complex and the von Hippel-Lindau (VHL) tumor suppressor complex. A major goal of the proposed research career is to understand how the ROC 1-dependent E3 ligases regulate the abundance of protein substrates required to control the progression of the cell cycle, the activation of signal transduction pathways and the execution of tumor suppressor activities. The initial studies have shown that ROC1-CUL1 functions as a core ubiquitin ligase and is recruited by an adapter, Skpl, and an F-box protein, HOS (beta-TRCP homologue), to form a four-subunit SCFHos/beta-Trcp-ROC1 complex that initiates and catalyzes the ubiquitination of phosphorylated IKB-alpha in the presence of Cdc34. These results have provided a conceptual molecular framework depicting the action of the ROC/APC11-CUL/APC2 ligases: a substrate targeting molecule, via an adapter protein, recruits a cognate core ligase element to promote the ubiquitination of its sequestered substrate. In this application, the principal investigator intends to continue our investigation focusing on the elucidation of the mechanisms by which the ROC 1-dependent E3 ligases engage to promote ubiquitination reactions. Specifically Dr. Pan proposes to: i) determine the mechanism of ROC 1-mediated Ubiquitin ligation reaction; ii) define the mechanism underlying the activation of the ROC 1-CULl ubiquitin ligase by Nedd8 conjugation; and iii) determine and characterize role of the novel F-box protein, Fbx22p44 in targeting KLF6, a transcription factor induced in response to liver injury, for ubiquitination. These proposed studies will provide information that: i) advances our knowledge of the mechanism of action of the ROC1 RING-H2 finger ubiquitin ligase; and ii) identify regulatory mechanisms governing the liver injury and recovery pathways.